Project Summary Mucosal associated invariant T (MAIT) cells, a subset of T cells restricted by MR1 (MR1T cells), are an innate- like T cell subset prevalent in humans and distributed throughout mucosal sites. Human MAIT cells are defined by the expression of the semi-invariant TCR? chain TRAV1-2/TRAJ12/20/33, restriction by the non- polymorphic major histocompatibility complex (MHC) class I-like molecule, MHC-related protein 1 (MR1), and by recognition of small organic molecules, derived from riboflavin biosynthesis. We have found that MAIT cells are both ?innate? effectors as evidenced by their functionality in the thymus, and have the capacity to adapt to their environment as evidenced by their effector memory cell surface phenotype and selective TCR usage. However, it is not clear if these expansions are driven by ongoing microbial or environmental exposures, or if MAIT cells retain the capacity to respond selectively to future antigenic challenges. If MAIT cells have memory, then they could be harnessed in future vaccination strategies. We provide evidence demonstrating oligoclonal expansions of pro-inflammatory MAIT cells in the human airway of subjects infected with TB. We also provide evidence that vaccination with BCG can result in functional MAIT cell expansion, and provide evidence that MAIT cells have an anti-microbial, polycytotoxic phenotype. We postulate that MR1T cells can facilitate the control of infection with Mtb, and will explore whether or not these cells have immunologic memory. Specific Aim 1: To determine whether or not CD8+ MR1T cells have features of immunologic memory. By determining the TCR repertoire of MR1T cells prior to antigenic exposure (human cord blood mononuclear cells; CBMC), following antigenic exposure as a result of infant vaccination with BCG, in the setting of TB (adult PBMC), in the lungs (BAL) and peripheral blood of subjects with pulmonary TB at the time of diagnosis and following treatment, we will establish if MAIT cells demonstrate TCR usage that reflects antigenic exposure, persistence following antigenic exposure, and dependence on the presence of antigen. Specifically, we anticipate that we will observe a broad repertoire of MR1T cell TCRs in human cord blood, and that in adults we will observe a narrower repertoire characterized by oligocolonal expansions. Following vaccination with BCG and in TB, we would expect to observe oligoclonal expansions in the PBMC and BAL, and would predict that these cells will return to the circulation following treatment. Finally, we will ask if these TCRs are associated with discrete ligand recognition. Specific Aim 2: To define the phenotype and functional capacity of MR1T cells. A combination of flow cytometric analysis of both ex vivo MR1T cells and MR1T cell clones, expression data analysis of ex vivo MR1T cells, and mechanistic evaluation of MR1T cell clones derived from each of the cohorts and time points described will be used to define the full functional and anti-microbial phenotype of these cells.